1. Field of the Invention
The present invention relates to gene constructs useful for modifying cell lines to effectuate glucose-regulated production of human insulin, and cell lines transformed by such gene constructs.
2. Background of the Related Art
Diabetes mellitus is a chronic disorder of fat, carbohydrate, and protein metabolism. It is characterized by an under-utilization of glucose and an absolute or relative insulin deficiency. Persons suffering from the disease in full expression have a tendency to fasting hyperglycemia, glycosuria, and ultimately to the development of atherosclerosis, neuropathy, nephropathy and microangiopathy. Diabetes is the seventh leading cause of death (the sixth-leading cause of death by disease) in the United States. It is also the leading cause of new cases of blindness in persons ages 20–74 and the leading cause of end-stage renal disease (accounting for about 40% of new cases). Persons afflicted with diabetes have about 15–50 times greater risk of leg amputation, 2–4 times the risk of heart disease, and are 2–4 times more likely to suffer a stroke than the general population.
The pathology of diabetes mellitus can be attributed to three major effects of insulin insufficiency: (1) a decrease in the utilization of glucose by the body cells, causing an increase in blood glucose to supranormal levels; (2) a marked increase in the mobilization of fats from the fat storage areas, causing abnormal fat metabolism as well as the deposition of lipids in vascular walls to cause atherosclerosis and (3) depletion of protein in the tissues of the body. Diabetes is a chronic disease that presently has no cure.
Diabetes mellitus is generally classified into three major forms: type 1 insulin-dependent diabetes (IDDM), often referred to as immune-mediated diabetes; type 2 non-insulin dependent diabetes (NIDDM); and diabetes due to variants in genes controlling beta cell function or metabolism. IDDM accounts for about 10% of all cases of diabetes. Type 1-IDDM is an immune-mediated disease that is associated with a near complete loss of the pancreatic beta cells resulting in insulin dependence for life. It can occur at any age, and it is estimated that about 1% of all newborns will develop the disease during their lifetime. Type 2-NIDDM accounts for most of the remaining 90% of cases of diabetes. Type 2-NIDDM is thought to occur as a result of both external and complex genetic influences which contribute to a peripheral insulin resistance with tissues failing to utilize glucose appropriately in response to the insulin signal. Allelic variants at the insulin locus itself have been associated with NIDDM, with the variants exhibiting altered properties with regard to transcriptional regulation. A very small percentage of diabetics have their condition secondary to variants in genes controlling beta cell function or metabolism.
Glucose homeostasis involves numerous neuroendocrine systems, however, the pancreatic islets of Langerhans are considered to be the primary “glucose sensor” in mammals. Pancreatic islets are composed of primarily four different populations of cells which may be characterized by their production of insulin, glucagon, somatostatin or pancreatic polypeptide. Insulin-producing beta cells predominate in the islets. However, the islet cell mass makes up only a small fraction (approximately 1%) of the total pancreas. Early stage Type 2-IDDM and Type 1-IDDM is characterized by a progressive loss of beta-cell function. Insulin secretion from islet beta-cells is stimulated by amino acids, three carbon sugars such as glyceraldehyde, and most prominently, glucose.
Transport of glucose into the beta cell, and metabolism of glucose therein, are absolute requirements for the secretion of insulin, i.e., (glucose stimulates insulin secretion from the beta-cells of the islets of Langerhans through its own metabolism). As in normal beta-cells glucose transport capacity is in excess relative to glycolytic flux, breakdown of glucose is the true rate-limiting step.
Glucose stimulates de novo insulin biosynthesis by increasing transcription, mRNA stability, translation and protein processing. It also rapidly stimulates the release of pre-stored insulin. The human insulin gene is encoded on the short arm of chromosome 11 and contains three exons and two introns. Transcriptional control of the insulin gene is achieved through a short region of flanking DNA that interacts with cell-specific and glucose-sensitive signaling molecules. The precise nature of the regulatory organization is poorly understood. However, it is generally believed that the basic helix-loop-helix and homeodomain-containing factors are critical components of the transcriptional machinery that governs beta-cell specific expression of insulin. The primary messenger RNA transcript is processed by the removal of intervening sequences and by the addition of a poly-A tail at its 3′ end to produce mature insulin messenger RNA. Translation of the m-RNA is at the rough endoplasmic reticulum yielding preproinsulin.
Preproinsulin differs from mature insulin in two ways: (1) it has a N-terminal “signal” or “pre” sequence which directs the polypeptide to the rough endoplasmic reticulum where it is proteolytically processed, and (2) contains an additional connecting peptide, known as the C-peptide, between the A and B chains, the C-peptide permitting correct folding of the whole molecule.
The signal sequence of preproinsulin is co-translationally removed in the endoplasmic reticulum to form proinsulin. Proinsulin is trafficked to the Golgi apparatus where it is subsequently transferred to regulated secretory vesicles wherein it is processed into mature insulin by proteolytic cleavage of the C-peptide.
Proinsulin processing occurs primarily in clathrin-coated immature secretory granules derived from the trans-Golgi network (TGN) of the pancreatic beta-cell. Proteolytic processing of proinsulin to insulin is by means of prohormone convertases of the subtilisin family of endoproteases. These convertases cleave peptide bonds carboxyl-terminal to dibasic residues, and in combination with carboxypeptidase E/H which removes the basic residues from the B chain, generating the mature functional insulin that is secreted into the circulation. That is, complete conversion involves endoproteolytic cleavage of the COOH-terminal to two pairs of basic residues linking the insulin B-chain to C-peptide (Arg31-Arg32) and C-peptide to the A-chain (Lys64-Arg65) as well as trimming of the remaining COOH-terminal basic residues by carboxypeptidase E/H (Davidson et al., Biochem. J. 245: 575 (1987); Grimwood et al., J. Biol. Chem. 264: 15662 (1989)). Secretory vesicles are derived from Golgi membranes by a process of budding off and eventual separation. The vesicles are transported to the plasma membrane surface of a cell in response to secretory stimuli such as glucose, where they fuse with the plasma membrane and release their stores of the mature hormone.
It has been hypothesized that glucose's specific stimulatory effect is mediated by, and proportional to, its flux rate through glycolysis and related pathways. Evidence is accumulating implicating a specific facilitated-diffusion type glucose transporter known as GLUT-2, and the glucose phosphorylating enzyme, glucokinase, in the control of glucose metabolism in islet beta-cells. Both proteins have affinities for glucose that allow large changes in activities over the physiological range of glucose.
Diabetes is treated by correcting insulin concentrations in the body in such a manner that the patient has normal or nearly normal carbohydrate, fat and protein metabolism as possible. Optimal therapy has been found to be effective at preventing most acute effects of diabetes, and to greatly delay the chronic effects as well. It is generally agreed that very little progress has been made in the clinical treatment of diabetic patients in the last twenty years.
Treatment for diabetes is still centered around self-injection of exogenous insulin once or twice daily, or in the case of non-severe diabetes wherein the islets still maintain the potential to secrete insulin, the use of drugs that stimulate insulin secretion such as the sulfonylureas. Exogenous insulin may be isolated by non-recombinant methods as from the purification of insulin from freshly isolated porcine or bovine pancreas, or by employment of recombination techniques. Recombinant methods generally include the expression of recombinant proinsulin in bacteria or yeast, followed by chemical treatment of the proinsulin to ensure correct disulfide bond linkages between the A and B chains of the mature insulin molecule. The mature insulin peptide is purified away from the bacterial or yeast proteins, as well as any added material. The bacterial recombinant procedure typically entails as many as forty distinct steps.
Treatment of diabetes conventionally also involves establishing the patient on a standard diet containing normal, well-controlled amounts of carbohydrates, and on a regular exercise program. Weight management is often useful as decreased fat reduces the insulin requirements of an individual. Exercise increases the transport of glucose into the muscle cells even in the absence of insulin, and thus actually has an insulin-like effect.
In conjunction with injections of insulin, diet, and exercise programs, the treatment of diabetes requires a constant and life-long monitoring of blood glucose. As many diabetic patients have difficulty in meeting all these strictures, they constantly expose themselves to the adverse effects of hypoglycemia and hyperglycemia. There is a need therefore for alternative methods for controlling blood glucose levels in the diabetic patient.
Numerous researchers have attempted to control insulin delivery through the use of external devices such as insulin pumps and pens. Unfortunately, such technology has not been developed to a point to permit tightly controlled blood glucose levels. Inadequacy of the blood glucose sensors, as well as the dispensing mechanism, plague currently available automated insulin dispensers.
Others have attempted to control blood glucose levels in the diabetic patient by transplantation of pancreatic tissue from a donor to the diabetic patient. Major problems associated with such transplantations include: the shortage of donor tissue, the cost and expense involved in harvesting donor tissue, the need for immunosuppression to prevent tissue rejection in non-isograft transplantations, and the difficulty in maintaining viable tissue for prolonged periods of time after harvest. Even successful transplants suffer from the inherent autoimmune mechanism responsible for destruction of the patient's original islet beta-cells.
An alternate approach that has been suggested entails the use of a biohybrid perfused “artificial pancreas” comprised of islet tissue in a selectively permeable membrane. The selectively permeable membrane acts to protect the transplanted islets from being recognized and destroyed by the same autoimmune mechanism responsible for the destruction of the patient's original beta-cells. The in vivo treatment of diabetes with peritoneal implants of encapsulated islets has been reported by several research groups (See, e.g., U.S. Pat. No. 5,262,055 to Bae et al. (1993); U.S. Pat. No. 5,427,940 to Newgard (1992); Lum et al., Diabetes 40: 1511 (1991); Maki et al., Transplantation 51: 43 (1991); Robertson, Diabetes 40: 1085 (1991); Colton et al., J. Biomech. Eng. 113: 152 (1991); Scharp et al., Diabetes 39: 515 (1990); Reach, Intern. J. Art. Organs 13: 329 (1990)). Such artificial pancreases are expensive and time-consuming to fabricate, and have been found to exhibit limited usefulness in practice.
A considerable amount of research has also been undertaken with respect to the transplantation of beta-cell cell lines. Beta-cell cell lines typically have been generated from insulinomas and hyperplastic islets. Two main approaches have been used to isolate immortalized beta-cell lines: (1) isolating the cells from an X-ray induced rat insulinoma; (2) infection and transformation of a primary culture of islet cells by simian virus 40 (SV40). Several of these cell lines display insulin secretion characteristics similar to those observed in intact adult islets, in particular the response to glucose concentrations in the physiological range (5–15 mmol/L). A common problem associated with such cells lines is their phenotypic instability. That is, after propagation of the cells in culture, the cells frequently become responsive to subphysiological concentrations of glucose and/or manifest diminished insulin output.
As cells from immortalized cell lines are subject to the same autoimmunity that destroyed the host's beta-cells, such beta cell lines are commonly encapsulated for transplantation. While encapsulation reduces immunological response to the cells, because the cells themselves are undergoing rapid cell division, the increasing oxygen and nutrient demand within the encapsulation, as well as the increase in metabolic wastes, adversely impact the survivability of the cells.
Several researchers have proposed genetically altering immortalized beta cells to control proliferation. For example, U.S. Pat. No. 6,114,599 to Erfat discloses immortalized beta cells genetically engineered such that proliferation of the cells is regulated by the presence or absence of an antibiotic. Such cells are produced by introducing into the beta cell, a first gene comprising a DNA encoding a TetR-VP16 gene fusion protein, and an insulin promoter which controls expression of the fusion protein, and a second gene comprising a DNA encoding SV40 T antigen, and a tetracycline operator minimal promoter. Such stable integration of both genes is achieved and screening is performed for cells whose proliferation is controlled by tetracycline or a derivative thereof. Erfat and coworkers have suggested that controlled oncogenesis might be used to amplify human islet cells in vitro, with subsequent transplantation of the islet cells after oncogene inactivation.
U.S. Pat. No. 5,723,333 to Levine et al. (Issued: Mar. 3, 1998), teaches pancreatic cell lines established by transforming cells with vectors, preferably retroviral vectors, containing two or more oncogenes under the control of one or more inducible promoters and/or genetic elements. A subpopulation of such cells was found to express high levels of insulin. PCT Publication WO 01/11031 to Giannoukakis et al. (Published: Feb. 15, 2001) discloses genetically engineered beta-cells comprising nucleic acid molecules encoding inhibitors of interleukin-1β (IL-1β) signal transduction. IL-1β has been shown to be the initiating cytokine that is directly responsible for the impairment of glucose-stimulated insulin production in human islets in vitro (MacDaniel et al., Proc. Soc. Exp. Biol. Med. 211: 24 (1996)). By reducing IL-1β activity there is a corresponding reduction in beta-cell dysfunction and apoptosis in the diabetic animal. Nucleic acid molecules encoding biologically active proteins capable of inhibiting IL-1β are said to include IL-1Ra, NF-Kβ inhibitor, AP1 inhibitor, soluble forms of the IL-1R, mutant forms of the fas or FADD protein, IGF-1, the cowpox crmA protein, and members of the bcl-2 family such as Bcl-2 and Bcl-XL.
A number of researchers have attempted to reverse diabetes by inducing embryonic stem cells to develop into insulin-producing cells. Embryonic stem cells are primitive cells that under appropriate direction can develop into any cell type. While it has been demonstrated that fetal pig pancreatic tissue is able to normalize blood glucose levels in a rat, such occurs only several months after the transplantation and requires immunosuppression of the rat.
U.S. Pat. No. 5,837,236 to Dinsmore (Issued: Nov. 17, 1998), teaches that the life of fetal porcine pancreatic cells can be extended in xenogenic subjects when cell-surface antigens capable of eliciting an immune response in the xenogenic subject is altered to a non-immunogenic antigen. Studies are underway to determine artificial conditions under which such cells will develop into insulin-producing cells. Such approach suffers from the cost associated with isolating embryonic stem cells, the problem of introducing cross-species viruses along with a transplantation of tissue from one species to another, and with respect to human stem cells, the moral implications raised with respect to the use of such cells.
Short et al., Am. J. Physiology 275: E748 (1998) disclose an embryonic kidney cell line transformed by introduction of a replication-defective adenovirus comprising human proinsulin cDNA in which the dibasic prohormone convertase recognition sequence was altered to a tetrabasic furin cleavage site. Furin is an endogenous protease present in the constitutive secretory pathway of many cells. Such cells were found to synthesize both proinsulin and mature insulin. Injection of the viral construct into the external jugular vein of mice resulted in insulin gene expression in the liver, but improvement in the glycemic state was transient lasting about two to three weeks.
Stewart et al., J. Mol. Endocrin. 11, 335 (1993) disclose transfection of a murine pituitary cell line with a human preproinsulin DNA in a plasmid containing a metallothionein promoter and a gene conferring resistance to the antibiotic G418. Such cells when implanted into non-diabetic athymic nude mice were found to delay streptozotocin-induced hyperglycemia (streptozotocin destroying beta-cells) compared to control mice receiving an implant of the non-transfected cells. The implanted cells however were seen to form a tumor-like aggregation.
Valera et al., FASEB J. 8: 440 (1994) demonstrated that hepatocytes could be modified to harbor the human insulin gene under the control of the phosphoenolpyruvate decarboxylase gene. Transgenic animals harboring such hepatocytes were seen to fare significantly better than non-transgenic controls in maintaining euglycemia when challenged with streptozotocin. Ectopic expression of insulin in hepatocytes of diabetic rats has also been reported by Kolodka et al. (Proc. Natl. Acad. Sci. 92: 3293 (1995) using in vivo retroviral gene transfer.
PCT Publication WO 97/14441 to Pollock et al. (Published: Apr. 24, 1997) teaches engineered cells expressing an exogenous or endogenous insulin gene in addition to an exogenous calbindin gene. Pollock et al. indicate that such cells exhibit the ability to secrete insulin in a glucose-sensitive fashion. They disclose that the cells may be enclosed in a semipermeable porous matrix which may be implanted into a diabetic animal to ameliorate insulin supplementation needs.
Sugiyama et al., Horm. Metab. Res. 29: 599 (1997), report transfection of hepatocytes with the rat insulin gene and lacZ using a defective adenoassociated viral (AAV) vehicle. Such cells are said to cause a reduction in glucose concentrations in surrounding medium and when transfected in vivo, a decrease in blood glucose levels Bartlett et al. (Transplantation Proceedings: 29, 2199 (1997)) disclose an expression vector which when injected directly into the hamstring muscle of normal or diabetic rats release proinsulin for up to 12 weeks. The vector comprises proinsulin genomic DNA inserted into the pAAV-CKM vector immediately after the CKM promoter.
U.S. Pat. No. 5,811,266 to Newgard (Issued: Sep. 22, 1998) discloses artificial beta-cells achieved through the introduction of one or more genes selected from the insulin gene, glucokinase gene, and glucose transporter gene, to provide an engineered cell having all three of the genes in a biologically functional and responsive configuration. Glucokinase, and the facilitated-diffusion type glucose transporter known as GLUT-2, are believed to be involved in the control of glucose metabolism in beta-cells.
PCT Publication WO 99/54451 to Powers et al. (Published: Oct. 28, 1999) teaches neuroendocrine cells secreting insulin. The engineered neuroendocrine cells which secrete insulin in response to glucose comprise a gene encoding a non-glucose insulin secretagogue receptor and an exogenous insulin gene. At least one of the genes is a recombinant gene introduced into the cell by means of a recombinant vector. It is hypothesized by the inventors that the ability of neuroendocrine cells to correctly process insulin reflects expression of similar hormone processing enzymes in a variety of neuroendocrine cells. The non-glucose insulin secretagogue receptors are said to include receptors for glucagon-like peptide 1, glucose-dependent insulin releasing polypeptide, cholecystokinin, gastrin, secretin, and gastric inhibitory peptide. Preferred cells for transformation are said to have an inherent capability of forming secretory granules, such as those from the pituitary and thyroid glands.
PCT Publication WO 00/31267 to Bosch et al. (Published: Jun. 2, 2000) teaches transfecting a precursor muscle cell or myoblast cell line with an exogenous gene encoding insulin. Preferred cell lines are myoblast cell lines like C2C12 which rapidly divide in the myoblast state and have the potential for differentiating into non-dividing myotubes. Such cells are said to be amenable to production in large quantities in the myoblast state, and after differentiation, capable of producing recombinant proteins with the use of a suitable promoter while not dividing. The promoter sequence is selected such that the promoter is activated or induced during diabetic conditions in the patient, and includes myosin light chain promoters, creatine kinase and myoD.
Lee et al., Nature 408: 483 (2000), report a recombinant adeno-associated virus (rAAV) capable of transforming a cell to express a single-chain insulin analogue (“SIA”), formed by replacing thirty-five residues of the C-peptide with a short turn-forming heptapeptide, which possess biologically-active insulin activity without enzymatic conversion, under the control of hepatocyte-specific L-type pyruvate kinase (LPK) promoter which regulates SIA expression in response to blood glucose levels. An albumin leader sequence was added to the SIA gene construct to facilitate the secretion of SIA from the cells, while the simian virus 40 enhancer (SV40) was added downstream of the SIA gene in order to elevate the basal level of SIA expression. The recombinant adeno-associated virus was found to cause the remission of diabetes in streptozotocin-induced diabetic rats and autoimmune diabetic mice for a prolonged time.
Barry et al., Human Gene Therapy 12: 131 (Jan. 20, 2001), have shown that retroviral vectors encoding glucose-responsive promoters driving furin expression may provide an amplified, glucose-regulated secretion of insulin. The group discloses a LhI*TFSN virus construct encoding a glucose-regulatable rat transforming growth factor α (TGFα) promoter controlling murine furin expression with a viral long terminal repeat promoter (LTR) driving constitutive expression of furin-cleavable human proinsulin. When such construct was transduced into vascular smooth muscles cells, the cells were seen to respond to physiological glucose concentrations. The furin-cleavable human proinsulin was obtained by altering human proinsulin cDNA to encode furin-cleavable sites (Hosaka et al., J. Biol. Chem. 255: 12127 (1991); Groskruetz et al., J. Biol. Chem. 269: 6241 (1994); Gros et al., Gene Ther. 8: 2249 (1997)). A selectable neo gene (bacterial neomycin phosophotransferase) was incorporated into the construct with the neo gene being expressed from and driven by the simian virus 40 promoter (SV40). Insulin release from the cells was adjudged against cells transduced with retroviral vectors comprising the LTR promoter driving either human adenosine deaminase cDNA or rat erythropoietin cDNA, and a SV40 promoter driving the selectable neo gene. Genetically altered cells were placed in a collagen matrix and implanted into a small pocket made in the stomach capsule of congenic DR lyp/lyp BB rats that had become diabetic (blood glucose exceeding 180 mg/dl). In eight treated rats, the researchers found a major reduction in insulin requirement to as low as 25% of pretreatment level for up to three months, with one rat becoming insulin free without hypoglycemia. Intraperitoneal glucose tolerance tests in diabetic rats receiving controlled cell implants did not show the characteristic decline in blood glucose of normal rats after glucose administration.
Numerous problems plague prior art “artificial beta-cells.” Such cells are typically very difficult to culture and are seen in practice to frequently loose their functional capabilities very quickly in culture. The majority of such cell lines either do not secrete sufficient insulin to be therapeutic or the insulin production is unregulated. In those cell lines that have shown some degree of glucose-regulated insulin production, most often a glucose responsive promoter transcriptionally regulates the insulin gene directly, failing to produce the C-peptide that has been reported to possess vasculature and neurologic functions. Transplantation of genetically-altered cells of the prior art typically also require the use of immune suppressing drugs for life, or incorporation into an immunoisolation device wherein prior art cells have frequently been found to be less than effective due to overgrowth or rapid senescence in the device.
There is a great need, therefore, for improved cell lines that are programmed to produce insulin in a glucose-responsive manner, that can produce mature insulin as well as the C-peptide that is reported to possess vasculature and neurologic functions, and that are adapted for growth to function over a significant period of time in an immunoisolation device so that no immunosuppression is required with respect to the transplant recipient.